This invention relates to the field of drug and xenobiotic metabolism. The invention includes novel cytochrome P450 fluorescent probe substrates and reaction products, methods for their preparation and their use as assay reagents.
Cytochromes P450 (CYP) are the principal enzymes for the oxidative metabolism of many drugs, procarcinogens, promutagens, and environmental pollutants. Cytochrome P450 is a heme-containing, membrane-bound, multienzyme system that is present in many tissues in vivo but is present at the highest level in liver. In human liver, it is estimated that there are 15-20 different xenobiotic-metabolizing cytochrome P450 forms. A standard nomenclature based on relatedness of amino acid sequences has been developed. A relatively limited subset of these enzymes, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 appear to be most commonly responsible for the metabolism of drugs and associated drug-drug interactions (Spatzenegger and Jaeger, 1995). The relative importance of this subset of enzymes is due to: the mass abundance of these enzymes (e.g. CYP3A4 is the most abundant P450 in human liver at xcx9c30% of total P450), the preference of these enzymes to bind and/or metabolize chemical structures commonly found in drugs (e.g. CYP2D6 preferentially binds and metabolizes drugs with basic amine functionalities), enzyme polymorphisms (such as CYP3A4 and CYP2C19), and enzyme regulation in response to environmental chemicals some (such as CYP1A2 and CYP3A4). Competition for metabolism by a particular cytochrome P450 form is a principal mechanism of some clinically significant drug-drug interactions.
Identification of the enzymes responsible for metabolism is becoming an important aspect of drug development. Such identifications consider both the metabolism of the new drug as well as inhibition by the new drug. The identification of enzymes involved in metabolism of the new drug allows prediction, based on knowledge of the ability of coadministered drugs to inhibit the same enzymes, of which coadministered drugs may inhibit the metabolism of the new drug. This information can also be used to predict individual variability based on known metabolic polymorphisms. The identification of the enzymes most sensitive to inhibition by the new drug allows prediction, based on knowledge of which coadministered drugs are metabolized by the same enzyme, of which coadministered drug""s metabolism may be inhibited by the new drug. Obtaining information for a series of drug candidates early in the drug discovery process can assist in the choice of the best drug candidate for further development.
Most cytochrome P450 assays have focused on the metabolism of drug molecules or drug candidates. While these chemicals are effective in assessing cytochrome P450 activity and inhibition, they are not amenable to high throughput screening assay technology (they require time consuming separation of enzyme reaction products using HPLC). Also, most of these substrates do not have the necessary fluorescent properties that make the substrate useful for in situ fluorescent plate analysis.
Sensitive fluorescent assays have been developed for human CYP1A1 and CYP1A2 enzymes. Members of the CYP1A family preferentially oxidize planar aromatic molecules not unlike the structure of common fluorescent chromophores. Microtiter plate based assays have been developed based on the O-dealkylation of alkyl ethers of resorufin (Donato, M. T. et. al., Anal. Biochem. 213, 29-33 (1993); Kennedy, S. W. and S. P. Jones, Anal. Biochem., 222, 217-223 (1994)) and coumarin (C. L. Crespi et al. Anal Biochem. 248, 188-190, (1997)). An intact cell assay using an alkyl ether of fluorescein (Quan, T. et al., Carcinogenesis 15, 1827-32, (1994)) has been described.
The development of fluorescent assays for other cytochromes P450 has been more challenging. We have described microtiter plate based assays for CYP2D6 based on alkylcoumarin derivatives (C. L. Crespi et al. Anal Biochem. 248, 188-190, (1997); Miller, V. P. and C. L. Crespi, U.S. patent application Ser. No. 09/352,576, entitled xe2x80x9cNovel CYP2D Fluorescent Assay Reagents,xe2x80x9d filed Jul. 12, 1999.) We have also reported the use of commercial alkylcoumarins for the fluorescent assay of CYP2C9 and CYP2C19. However, the sensitivity (signal to noise ratio) of these CYP2C assays is limited. New substrates which increase the assay signal and minimize the required amount of enzyme reagent would be useful. No fluorescent substrate has been reported for the important drug metabolizing enzyme CYP2C8. CYP2C8 is the primary cytochrome P450 enzyme responsible for the metabolism of the anticancer drug paclitaxel (TAXOL(trademark)).
Among the different cytochromes P450 expressed in the liver, CYP3A4 is the most abundant. Important classes of substrates for this enzyme include steroids, macrolide antibiotics, antivirals, and polycyclic aromatic hydrocarbons. As the majority of commercially available drugs are metabolized by CYP3A4, the importance of screening for inhibition of this enzyme is essential in drug development. We have previously reported the use of the commercially available 7-benzyloxyresorufin (BzRes) as a fluorescent substrate for assessing CYP3A4 activity in a high throughput mode (Crespi et al. Anal Biochem. 248, 188-190, (1997)). We and others have demonstrated that the inhibition potential (e.g. IC50 value) or activation potential for most compounds varies dramatically depending on the probe substrate used (K E Thummel and G R Wilkinson, Ann. Rev. Pharmacol. Toxicol. 38:389-430 (1998)). Additional fluorescent probe substrates, which differ significantly in structure and chemical properties from the current substrates, are needed to gain a better understanding of the potential for test compounds to inhibit CYP3A4.
Fluorescein ethers have been reported to be useful fluorescent cytochrome P450 assay reagents. A. G. Miller (A. G. Miller, Anal. Biochem. 133, 46-57 (1983)) described a series of ethylated fluoresceins. Ethoxyfluorescein ethyl ether, reportedly was the most useful for the flow cytometric analysis and sorting of intact, viable rat cells based on cytochrome P450 activity. Other laboratories have also reported using the diethoxy- or a dimethoxyflourescein derivative for the same purpose with rat or porcine cells (White et al., Biochem. J. 247, 23-28 (1987); Pan et al, Artif. Organs 20, 1173-1180 (1996); Anderson et al., Int. J. Artif. Organs 21, 360-364, (1998)). More recently, ethoxyfluorescein ethyl ester has been reported to monitor the activity of human skin fibroblasts transfected with human CYP1A1 cDNA (Quan, T. et al., Carcinogenesis 15, 1827-32, (1994)).
Aryl ether derivatives of fluorescein have not been reported as substrates for cytochrome P450 enzymes. A benzyl ether derivative of fluorescein, benzyloxyfluorescein benzyl ester, has been reported as an intermediate in the synthesis of fluorescein labeled polymers (Hargreaves and Webber, Can. J. Chem. 63, 1320-1327 (1985)), chiral prodyes (Tadic and Brossi, Heterocycles 31, 1975-1982, (1990)), and dye precursors (Dombrowski Jr. et al. U.S. Pat. No. 5,196,297).
The present invention relates to novel fluorescent substrates of cytochrome P450 enzymes. These substrates are useful in assessing cytochrome P450 enzyme activity and in selecting compounds which inhibit cytochrome P450 enzyme activity. Accordingly, the compounds and methods of the invention are useful for identifying potential adverse drug interactions which are mediated by inhibition of cytochrome P450 enzyme activity.
The compounds of the invention are substrates that are characterized in having properties which permit the sensitive quantitation of CYP2C8, CYP2C9, CYP2C19, and CYP3A activity using in situ fluorescence analysis. To satisfy these requirements, the compounds of the invention include: 1) An aryl ether group at position 6 of fluorescein which can be easily O-dealkylated by the enzyme, and 2) a fluorescein core for ease in fluorescence detection.
The enzyme reaction and assay method which describe the invention are illustrated in Reaction Scheme 1, below. Cytochrome P450 enzymes catalyze the dealkylation of compounds of Formula I to the intermediate of Formula II. Addition of base to the assay mixture hydrolyzes the ester intermediate (if present) to fluorescein which is quantitated spectrofluorometrically. 
According to one aspect of the invention, compounds of Formula A are provided:
Formula A: 
(a) wherein R1 and R2 are independently selected from the group consisting of an hydrido and an aryl, provided that R1 and R2 are not both hydrido; and wherein the aryl contains an aryl ring carbon and/or an aryl ring nitrogen and the (CH2)n or (CH2)m is coupled via a covalent bond to the aryl ring carbon or the aryl ring nitrogen;
(b) wherein n is 0, 1, 2, or 3;
(c) wherein X1, X2, X3, X4, X5, and X6 are independently selected from the group consisting of an hydrido, a chloro, a fluoro, a bromo, and an iodo, provided that when R1 and R2 are phenyl, and n and m are 1, at least one of X1, X2, X3, X4, X5, and X6 is not hydrido;
(d) wherein m is 0, 1, 2, or 3; and
(e) wherein the compound is a cytochrome P450 substrate or a cytochrome P450 reaction product.
The above structure embraces three preferred embodiments, which are referred to herein as: (1) compounds of Formula I; (2) compounds of Formula II; and (3) compounds of Formula III. Compounds of Formula I and III are substrates for cytochrome P450 enzymes; compounds of Formula II are the products of a cytochrome P450 reaction in which a compound of Formula I is the substrate, i.e., the compounds of Formula II can be produced by the process of contacting a compound of Formula I with a cytochrome P450 under conditions whereby the cytochrome P450 enzyme catalyzes the conversion of the substrate to the reaction product.
According to certain embodiments, compounds of Formula I are provided:
Formula I: 
(a) wherein R1 is aryl and R2 is independently selected from the group consisting of an hydrido and an aryl, provided that R1 and R2 are not both phenyl when n and m are 1; and wherein the aryl contains an aryl ring carbon and/or an aryl ring nitrogen and the (CH2)n or (CH2)m is coupled via a covalent bond to the aryl ring carbon or the aryl ring nitrogen;
(b) wherein n is 0, 1, 2, or 3;
(c) wherein X1, X2, X3, X4, X5, and X6 are independently selected from the group consisting of an hydrido, a chloro, a fluoro, a bromo, and an iodo;
(d) wherein m is 0, 1, 2, or 3; and
(e) wherein the compound is a cytochrome P450 substrate.
In the most preferred embodiments of the compound of Formula I, X2 and X5 are chloro and/or fluoro. In yet other embodiments, X2 and X5 may or may not be halogenated (particularly chloro and/or fluoro) and one or both of R1 and R2 are phenyl when one or both of n and m are 1. In still other preferred embodiments one or both of R1 and R2 are phenyl when one or both of n and m are 1, and the phenyl further includes (e.g., at the para position) an electron donating group, such as a halogen (especially chloro, fluoro), C (halogen)3, or NH3+.
According to yet other embodiments, compounds of Formula II are provided:
A3. The compound of claim A1, having Formula II: 
(a) wherein R1 is hydrido and R2 is an aryl; and wherein the aryl contains an aryl ring carbon and/or an aryl ring nitrogen and the (CH2)m is coupled via a covalent bond to the aryl ring carbon or the aryl ring nitrogen;
(b) wherein n is 0;
(c) wherein X1, X2, X3, X4, X5, and X6 are independently selected from the group consisting of an hydrido, a chloro, a fluoro, a bromo, and an iodo;
(d) wherein m is 0, 1, 2, or 3; and
(e) wherein the compound is a cytochrome P450 reaction product.
For the most preferred embodiments of the compound of Formula II, X2 and X5 are chloro and/or fluoro. In yet other embodiments, X2 and X5 may or may not be halogenated (particularly chloro and/or fluoro), R2 is phenyl and m is 1. In still other preferred embodiments R2 is phenyl, m is 1, and the phenyl further includes (e.g., at the para position) an electron donating group, such as a halogen (especially chloro, fluoro), C (halogen)3, or NH3+.
A particularly preferred embodiment of the compound of Formula II is compound IIa: 
Compound IIa
According to still other embodiments, compounds of Formula III are provided:
Formula III: 
(a) wherein R1 and R2 are each phenyl; and wherein the phenyl contains an aryl ring carbon and the (CH2)n or (CH2)m is coupled via a covalent bond to the aryl ring carbon;
(b) wherein n is 1;
(c) wherein X1, X2, X3, X4, X5, and X6 are independently selected from the group consisting of a chloro, a fluoro, a bromo, and an iodo;
(d) wherein m is 1; and
(e) wherein the compound is a cytochrome P450 substrate.
For the most preferred embodiments of the compound of Formula III, X2 and X5 are chloro and/or fluoro. In yet other embodiments, X2 and X5 may or may not be halogenated (particularly chloro and/or fluoro). In still other preferred embodiments R2 is phenyl and the phenyl further includes (e.g., at the para position) an electron donating group, such as a halogen (especially chloro, fluoro), C (halogen)3, or NH3+.
According to yet another aspect of the invention, a composition comprising a compound of Formula I, a compound of Formula II, a compound of Formula III, and/or Benzyloxyfluorescein benzyl ester (xe2x80x9cDBFxe2x80x9d; 97744-44-0; Benzoic acid, 2-[3-oxo-6-(phenylmethoxy)-3H-xanthen-9-yl)-benzyl ester (CA index name), Hargreaves, J. S. and S. E. Webber (1985) Can. J. Chem. 63, 1320-1327)) is provided. The compound is present in the composition at a concentration greater than at least 50% by weight. The most preferred compositions contain a concentration of the compound of Formula I, II, III, or DBF that is at least 80%, more preferably at least 90%, and most preferably at least 95% by weight. Preferably, the compositions are aqueous preparations which are free of contaminants that would interfere with a cytochrome P450 enzyme reaction. Certain embodiments of the preferred compositions are also substantially free of detectable reaction product, i.e., the compositions of the invention do not contain levels of the cytochrome P450-catalyzed reaction product. In these and other embodiments, the compositions optimally further contain a cytochrome P450, e.g., an isolated enzyme or a microsome expressing a cDNA expressed cytochrome P450.
The compositions may be contained in vials that are components of a kit for assaying cytochrome P450 enzyme activity. The vials may contain preselected amounts of the compositions to facilitate dissolution of the contents to achieve a preselected concentration of the compound for performing a cytochrome P450 enzyme assay. Accordingly, in certain embodiments of the invention, the compositions contain the appropriate buffers for performing an enzyme reaction in which the compound of Formula I, the compound of Formula III, or DBF serves as the substrate to form a fluorescent product.
According to yet another aspect of the invention, a method for assaying cytochrome P450 enzyme activity is provided. The method involves contacting a cytochrome P450 enzyme with a compound of Formula I, a compound of Formula III, or DBF under conditions whereby the cytochrome P450 enzyme catalyzes the conversion of the compound (substrate) to a cytochrome P450 reaction product. Such conditions are generally known to those of ordinary skill in the art and are illustrated in the Examples. 
For ease of illustration, the above reaction is shown with respect to substrates which are compounds of Formula I as defined herein. It is to be understood that the reaction also can be performed wherein the substrate is a compound of Formula III as defined herein or DBF and that these compounds optionally are halogenated as shown in their respective formulas.
The method for assaying cytochrome P450 enzyme activity is used to detect activity of a cytochrome P450 that may be contained in biological fluid sample or solid sample (e.g., a biopsy sample from liver, brain or intestine) or that may be expressed in a cell-containing or cell-free system (e.g., a microsome containing cDNA-expressed cytochrome P450). In this manner, conditions associated with deficiencies or over expression of cytochrome P450 enzyme activity can be detected.
The cytochrome P450 assay may be performed in vivo or in vitro. For example, the compounds of the invention (e.g., the compounds of Formula I, the compounds of Formula III, or DBF) can be administered to an animal model for, e.g., locating and, optionally, quantifying, cytochrome P450 enzyme activity (e.g., by observing reaction products in biological fluid or tissue samples of the animal). More preferably, the method for assaying cytochrome P450 enzyme activity is used to detect activity of a cytochrome P450 that may be contained in biological fluid sample or solid sample (e.g., a biopsy sample from liver, brain or intestine) or that may be expressed in a cell-containing or cell-free system (e.g., a microsome containing cDNA-expressed cytochrome P450). In this manner, conditions associated with deficiencies or over expression of cytochrome P450 enzyme activity can be detected. Thus, the cytochrome P450 enzyme may be contained in a sample that is a liver sample such as a crude homogenate, partially purified, or purified liver enzyme obtained from a biopsy, a cDNA-expressed cytochrome P450, in hepatocytes, or in microsomes.
According to yet another aspect of the invention, a screening method for identifying agents which inhibit cytochrome P450 enzyme activity is provided. The method involves contacting a cytochrome P450 enzyme with a compound of Formula I, a compound of Formula III , or DBF in the presence of a putative cytochrome P450 enzyme inhibitor and identifying an agent which inhibits the cytochrome P450 enzyme activity as the cytochrome P450 enzyme inhibitor. In the preferred embodiments, the screening method is a high throughput screening assay. More preferably, the screening assay is performed in a multiwell (e.g., microtiter) plate or a container for containing a relatively small volume, e.g., the compound of Formula I, Formula III, or DBF is contacted with the putative cytochrome P450 enzyme inhibitor and the cytochrome P450 enzyme in a microtiter plate well or a small vial. In certain embodiments, the compounds of Formula I, Formula III, or DBF can be provided in the microtiter plate well or small vial. For example, the compounds can be distributed into one or more vials, which are then lyophilized or otherwise dried to provide a product having an enhanced shelf life. If the product is provided for use in a kit for measuring cytochrome P450 enzyme activity, the kit can further contain instructions for redissolving the compounds of Formula I, Formula III, or DBF and, optionally, an appropriate buffer (e.g., enzyme reaction buffer) for effecting the dissolution.
According to another aspect of the invention, a method for visualizing a cytochrome P450 enzyme is provided. The method involves contacting a cytochrome P450 enzyme-containing sample with a compound of Formula I, a compound of Formula III, or DBF and subjecting the cytochrome P450 enzyme and the compound to conditions whereby the cytochrome P450 enzyme catalyzes the conversion of the compound of Formula I , the compound of Formula III, or DBF to a fluorescent product. In the preferred embodiments, the method for visualizing a cytochrome P450 enzyme is performed on a tissue section sample, i.e., the cytochrome P450 enzyme-containing sample is a tissue section such as derived from a biopsy sample.
According to still another aspect of the invention, kits for detecting and/or measuring cytochrome P450 enzyme activity are provided. The kits contain a compound of Formula I, Formula III, and/or DBF and instructions for using the kits to measure cytochrome P450 enzyme activity. The kits may further contain instructions for calculating Ki and/or IC50 for a cytochrome P450 inhibitor. The preferred compounds of Formula I and compounds of Formula III are compounds which have a high specificity of binding for the enzyme and for which the enzyme exhibits a high rate of substrate turnover. These parameters typically are reflected in the Km and Vmax values for the enzyme-catalyzed conversion of the substrate (i.e., compound of the invention) to a fluorescent product. In general, a higher relative affinity of the cytochrome P450 enzyme for a first substrate compared to a second substrate is indicated by a lower Km value for the first substrate compared to the second substrate. A higher catalytic turnover for a first substrate compared to a second substrate is indicated by a higher Vmax for the first substrate. The preferred compounds of the invention have a Km of greater than about 0.50 uM with a Vmax greater than about 0.05 minxe2x88x921. In general, the compounds of the invention have a Km from about 0.5 uM to about 5 uM and a Vmax from about 0.10 or from about 0.20 to about 50 minxe2x88x921, with a preferred range for Km being greater than about 0.50 uM and a preferred range for Vmax being from about 0.20 minxe2x88x921 to about 50 minxe2x88x921; with Vmax being more preferably from about 2.0 or from about 20.0 to about 50 minxe2x88x921.
According to yet another aspect of the invention, novel cytochrome P450 fluorescent products are provided. The novel products are compounds of Formula A wherein R1 is hydrido and n is 0 (see Formula II). In general, these novel products are produced as a reaction product of the cytochrome P450-catalyzed reaction of a substrate that is a compound of Formula I, a compound of Formula III, or DBF. In general, these compounds have structures that differ from those of the compounds of Formula I, Formula III, and DBF in having a hydroxy group at position 6 of the fluorescein.
These and other aspects of the invention as well as various advantages and utilities will be more apparent with reference to the detailed description of the preferred embodiments. All patents, patent publications and references identified in this document are incorporated in their entirety herein by reference.